Biopolymer marker indicative of disease state having a molecular weight of 1518 daltons

ABSTRACT

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to evidence at least one particular disease state; thereby enabling a diagnostician to gain the ability to characterize either the presence or absence of said at least one disease state relative to recognition of the presence and/or the absence of said biopolymer.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of application serialnumber 09/845,765 filed on Apr. 30, 2001, the contents of which isherein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the field of characterizing the existence of adisease state; particularly to the utilization of mass spectroscopy toelucidate particular biopolymer markers indicative of disease state, andmost particularly to specific biopolymer sequences having a uniquerelationship to at least one particular disease state.

BACKGROUND OF THE INVENTION

Methods utilizing mass spectrometry for the analysis of a targetpolypeptide have been taught wherein the polypeptide is firstsolubilized in an appropriate solution or reagent system. The type ofsolution or reagent system, e.g., comprising an organic or inorganicsolvent, will depend on the properties of the polypeptide and the typeof mass spectrometry performed and are well known in the art (see, e.g.,Vorm et al. (1994) Anal. Chem. 66:3281 (for MALDI) and Valaskovic et al.(1995) Anal. Chem. 67:3802 (for ESI). Mass spectrometry of peptides isfurther disclosed, e.g., in WO 93/24834 by Chait et al.

In one prior art embodiment, the solvent is chosen so that the risk thatthe molecules may be decomposed by the energy introduced for thevaporization process is considerably reduced, or even fully excluded.This can be achieved by embedding the sample in a matrix, which can bean organic compound, e.g., sugar, in particular pentose or hexose, butalso polysaccharides such as cellulose. These compounds are decomposedthermolytically into CO₂ and H₂O so that no residues are formed whichmight lead to chemical reactions. The matrix can also be an inorganiccompound, e.g., nitrate of ammonium which is decomposed practicallywithout leaving any residues. Use of these and other solvents arefurther disclosed in U.S. Pat. No. 5,062,935 by Schlag et al.

Prior art mass spectrometer formats for use in analyzing the translationproducts include ionization (I) techniques, including but not limited tomatrix assisted laser desorption (MALDI), continuous or pulsedelectrospray (ESI) and related methods (e.g., IONSPRAY or THERMOSPRAY),or massive cluster impact (MCI); these ion sources can be matched withdetection formats including linear or non-linear reflectiontime-of-flight (TOF), single or multiple quadropole, single or multiplemagnetic sector, Fourier Transform ion cyclotron resonance (FTICR), iontrap, and combinations thereof (e.g., ion-trap/time-of-flight). Forionization, numerous matrix/wavelength combinations (MALDI) or solventcombinations (ESI) can be employed. Subattomole levels of protein havebeen detected, for example, using ESI (Valaskovic, G. A. et al., (1996)Science 273:1199-1202) or MALDI (Li, L. et al., (1996) J. Am. Chem. Soc.118:1662-1663) mass spectrometry.

ES mass spectrometry has been introduced by Fenn et al. (J. Phys. Chem.88, 4451-59 (1984); PCT Application No. WO 90/14148) and currentapplications are summarized in recent review articles (R. D. Smith etal., Anal. Chem. 62, 882-89 (1990) and B. Ardrey, Electrospray MassSpectrometry, Spectroscopy Europe, 4, 10-18 (1992)). MALDI-TOF massspectrometry has been introduced by Hillenkamp et al. (“Matrix AssistedUV-Laser Desorption/Ionization: A New Approach to Mass Spectrometry of XLarge Biomolecules,” Biological Mass Spectrometry (Burlingame andMcCloskey, editors), Elsevier Science Publishers, Amsterdam, pp. 49-60,1990). With ESI, the determination of molecular weights in femtomoleamounts of sample is very accurate due to the presence of multiple ionpeaks which all could be used for the mass calculation.

The mass of the target polypeptide determined by mass spectrometry isthen compared to the mass of a reference polypeptide of known identity.In one embodiment, the target polypeptide is a polypeptide containing anumber of repeated amino acids directly correlated to the number oftrinucleotide repeats transcribed/translated from DNA; from its massalone the number of repeated trinucleotide repeats in the original DNAwhich coded it, may be deduced.

U.S. Pat. No. 6,020,208 utilizes a general category of probe elements(i.e., sample presenting means) with Surfaces Enhanced for LaserDesorption/Ionization (SELDI), within which there are three (3) separatesubcategories. The SELDI process is directed toward a sample presentingmeans (i.e., probe element surface) with surface-associated (orsurface-bound) molecules to promote the attachment (tethering oranchoring) and subsequent detachment of tethered analyte molecules in alight-dependent manner, wherein the said surface molecule(s) areselected from the group consisting of photoactive (photolabile)molecules that participate in the binding (docking, tethering, orcrosslinking) of the analyte molecules to the sample presenting means(by covalent attachment mechanisms or otherwise).

PCT/EP/04396 teaches a process for determining the status of an organismby peptide measurement. The reference teaches the measurement ofpeptides in a sample of the organism which contains both high and lowmolecular weight peptides and acts as an indicator of the organism'sstatus. The reference concentrates on the measurement of low molecularweight peptides, i.e. below 30,000 Daltons, whose distribution serves asa representative cross-section of defined controls. Contrary to themethodology of the instant invention, the '396 patent strives todetermine the status of a healthy organism, i.e. a “normal” and then usethis as a reference to differentiate disease states. The presentinventors do not attempt to develop a reference “normal”, but ratherstrive to specify particular markers which are evidentiary of at leastone specific disease state, whereby the presence of said marker servesas a positive indicator of disease. This leads to a simple method ofanalysis which can easily be performed by an untrained individual, sincethere is a positive correlation of data. On the contrary, the '396patent requires a complicated analysis by a highly trained individual todetermine disease state versus the perception of non-disease or normalphysiology.

Richter et al, Journal of Chromatography B, 726(1999) 25-35, refer to adatabase established from human hemofiltrate comprised of a massdatabase and a sequence database. The goal of Richter et al was toanalyze the composition of the peptide fraction in human blood. UsingMALDI-TOF, over 20,000 molecular masses were detected representing anestimated 5,000 different peptides. The conclusion of the study was thatthe hemofiltrate (HF) represented the peptide composition of plasma. Nocorrelation of peptides with relation to normal and/or disease states ismade.

As used herein, “analyte” refers to any atom and/or molecule; includingtheir complexes and fragment ions. In the case of biologicalmolecules/macromolecules or “biopolymers”, such analytes include but arenot limited to: proteins, peptides, DNA, RNA, carbohydrates, steroids,and lipids. Note that most important biomolecules under investigationfor their involvement in the structure or regulation of life processesare quite large (typically several thousand times larger than H₂O.

As used herein, the term “molecular ions” refers to molecules in thecharged or ionized state, typically by the addition or loss of one ormore protons (H⁺)

As used herein, the term “molecular fragmentation” or “fragment ions”refers to breakdown products of analyte molecules caused, for example,during laser-induced desorption (especially in the absence of addedmatrix).

As used herein, the term “solid phase” refers to the condition of beingin the solid state, for example, on the probe element surface.

As used herein, “gas” or “vapor phase” refers to molecules in thegaseous state (i.e., in vacuo for mass spectrometry).

As used herein, the term “analyte desorption/ionization” refers to thetransition of analytes from the solid phase to the gas phase as ions.Note that the successful desorption/ionization of large, intactmolecular ions by laser desorption is relatively recent (circa 1988)—thebig breakthrough was the chance discovery of an appropriate matrix(nicotinic acid).

As used herein, the term “gas phase molecular ions” refers to those ionsthat enter into the gas phase. Note that large molecular mass ions suchas proteins (typical mass=60,000 to 70,000 times the mass of a singleproton) are typically not volatile (i.e., they do not normally enterinto the gas or vapor phase). However, in the procedure of the presentinvention, large molecular mass ions such as proteins do enter the gasor vapor phase.

As used herein in the case of MALDI, the term “matrix” refers to any oneof several small, acidic, light absorbing chemicals (e.g., nicotinic orsinapinic acid) that is mixed in solution with the analyte in such amanner so that, upon drying on the probe element, the crystallinematrix-embedded analyte molecules are successfully desorbed (by laserirradiation) and ionized from the solid phase (crystals) into thegaseous or vapor phase and accelerated as intact molecular ions. For theMALDI process to be successful, analyte is mixed with a freshly preparedsolution of the chemical matrix (e.g., 10,000:1 matrix:analyte) andplaced on the inert probe element surface to air dry just before themass spectrometric analysis. The large fold molar excess of matrix,present at concentrations near saturation, facilitates crystal formationand entrapment of analyte.

As used herein, “energy absorbing molecules (EAM)” refers to any one ofseveral small, light absorbing chemicals that, when presented on thesurface of a probe, facilitate the neat desorption of molecules from thesolid phase (i.e., surface) into the gaseous or vapor phase forsubsequent acceleration as intact molecular ions. The term EAM ispreferred, especially in reference to SELDI. Note that analytedesorption by the SELDI process is defined as a surface-dependentprocess (i.e., neat analyte is placed on a surface composed of boundEAM). In contrast, MALDI is presently thought to facilitate analytedesorption by a volcanic eruption-type process that “throws” the entiresurface into the gas phase. Furthermore, note that some EAM when used asfree chemicals to embed analyte molecules as described for the MALDIprocess will not work (i.e., they do not promote molecular desorption,thus they are not suitable matrix molecules).

As used herein, “probe element” or “sample presenting device” refers toan element having the following properties: it is inert (for example,typically stainless steel) and active (probe elements with surfacesenhanced to contain EAM and/or molecular capture devices).

As used herein, “MALDI” refers to Matrix-Assisted LaserDesorption/Ionization.

As used herein, “TOF” stands for Time-of-Flight.

As used herein, “MS” refers to Mass Spectrometry.

As used herein “MALDI-TOF MS” refers to Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

As used herein, “ESI” is an abbreviation for Electrospray ionization.

As used herein, “chemical bonds” is used simply as an attempt todistinguish a rational, deliberate, and knowledgeable manipulation ofknown classes of chemical interactions from the poorly defined kind ofgeneral adherence observed when one chemical substance (e.g., matrix) isplaced on another substance (e.g., an inert probe element surface).Types of defined chemical bonds include electrostatic or ionic (±) bonds(e.g., between a positively and negatively charged groups on a proteinsurface), covalent bonds (very strong or “permanent” bonds resultingfrom true electron sharing) coordinate covalent bonds (e.g., betweenelectron donor groups in proteins and transition metal ions such ascopper or iron), and hydrophobic interactions (such as between twononcharged groups).

As used herein, “electron donor groups” refers to the case ofbiochemistry, where atoms in biomolecules (e.g, N, S, O) “donate” orshare electrons with electron poor groups (e.g., Cu ions and othertransition metal ions).

With the advent of mass spectroscopic methods such as MALDI and SELDI,researchers have begun to utilize a tool that holds the promise ofuncovering countless biopolymers which result from translation,transcription and post-translational transcription of proteins from theentire genome.

Operating upon the principles of retentate chromatography, SELDI MSinvolves the adsorption of proteins, based upon their physico-chemicalproperties at a given pH and salt concentration, followed by selectivelydesorbing proteins from the surface by varying pH, salt, or organicsolvent concentration. After selective desorption, the proteins retainedon the SELDI surface, the “chip”, can be analyzed using the CIPHERGENprotein detection system, or an equivalent thereof. Retentatechromatography is limited, however, by the fact that if unfractionatedbody fluids, e.g. blood, blood products, urine, saliva, and the like,along with tissue samples, are applied to the adsorbent surfaces, thebiopolymers present in the greatest abundance will compete for all theavailable binding sites and thereby prevent or preclude less abundantbiopolymers from interacting with them, thereby reducing or eliminatingthe diversity of biopolymers which are readily ascertainable.

If a process could be devised for maximizing the diversity ofbiopolymers discernable from a sample, the ability of researchers toaccurately determine the relevance of such biopolymers with relation toone or more disease states would be immeasurably enhanced.

SUMMARY OF THE INVENTION

The instant invention is characterized by the use of a combination ofpreparatory steps in conjunction with SELDI mass spectroscopy andtime-of-flight detection procedures to maximize the diversity ofbiopolymers which are verifiable within a particular sample. The cohortof biopolymers verified within a sample is then viewed with reference totheir ability to evidence at least one particular disease state; therebyenabling a diagnostician to gain the ability to characterize either thepresence or absence of said at least one disease state relative torecognition of the presence and/or the absence of said biopolymer.

Although all manner of biomarkers related to all disease conditions aredeemed to be within the purview of the instant invention andmethodology, particular significance was given to those markers anddiseases associated with the complement system and Syndrome X anddiseases related thereto.

The complement system is an important part of non-clonal or innateimmunity that collaborates with acquired immunity to destroy invadingpathogens and to facilitate the clearance of immune complexes from thesystem. This system is the major effector of the humoral branch of theimmune system, consisting of nearly 30 serum and membrane proteins. Theproteins and glycoproteins composing the complement system aresynthesized largely by liver hepatocytes. Activation of the complementsystem involves a sequential enzyme cascade in which the proenzymeproduct of one step becomes the enzyme catalyst of the next step.Complement activation can occur via two pathways: the classical and thealternative. The classical pathway is commonly initiated by theformation of soluble antigen-antibody complexes or by the binding ofantibody to antigen on a suitable target, such as a bacterial cell. Thealternative pathway is generally initiated by various cell-surfaceconstituents that are foreign to the host. Each complement component isdesignated by numerals (C1-C9), by letter symbols, or by trivial names.After a component is activated, the peptide fragments are denoted bysmall letters. The complement fragments interact with one another toform functional complexes. Ultimately, foreign cells are destroyedthrough the process of a membrane-attack complex mediated lysis.

The C4 component of the complement system is involved in the classicalactivation pathway. It is a glycoprotein containing three polypeptidechains (α, β, and γ). C4 is a substrate of component C1s and isactivated when C1s hydrolyzes a small fragment (C4a) from the aminoterminus of the α chain, exposing a binding site on the larger fragment(C4b).

The native C3 component consists of two polypeptide chains, α and β. Asa serum protein, C3 is involved in the alternative pathway. Serum C3,which contains an unstable thioester bond, is subject to slowspontaneous hydrolysis into C3a and C3b. The C3f component is involvedin the regulation required of the complement system which confines thereaction to designated targets. During the regulation process, C3b iscleaved into two parts: C3bi and C3f. C3bi is a membrane-boundintermediate wherein C3f is a free diffusible (soluble) component.

Complement components have been implicated in the pathogenesis ofseveral disease conditions. C3 deficiencies have the most severeclinical manifestations, such as recurrent bacterial infections andimmune-complex diseases, reflecting the central role of C3. The rapidprofusion of C3f moieties and resultant “accidental” lysis of normalcells mediated thereby gives rise to a host of auto-immune reactions.The ability to understand and control these mechanisms, along with theirattendant consequences, will enable practitioners to develop bothdiagnostic and therapeutic avenues by which to thwart these maladies.

In the course of defining a plurality of disease specific markersequences, special significance was given to markers which wereevidentiary of a particular disease state or with conditions associatedwith Syndrome-X. Syndrome-X is a multifaceted syndrome, which occursfrequently in the general population. A large segment of the adultpopulation of industrialized countries develops this metabolic syndrome,produced by genetic, hormonal and lifestyle factors such as obesity,physical inactivity and certain nutrient excesses. This disease ischaracterized by the clustering of insulin resistance andhyperinsulinemia, and is often associated with dyslipidemia (atherogenicplasma lipid profile), essential hypertension, abdominal (visceral)obesity, glucose intolerance or noninsulin-dependent diabetes mellitusand an increased risk of cardiovascular events. Abnormalities of bloodcoagulation (higher plasminogen activator inhibitor type I andfibrinogen levels), hyperuricemia and microalbuminuria have also beenfound in metabolic syndrome-X.

The instant inventors view the Syndrome X continuum in itscardiovascular light, while acknowledging its important metaboliccomponent. The first stage of Syndrome X consists of insulin resistance,abnormal blood lipids (cholesterol and triglycerides), obesity, and highblood pressure (hypertension). Any one of these four first stageconditions signals the start of Syndrome X.

Each first stage Syndrome X condition risks leading to another. Forexample, increased insulin production is associated with high blood fatlevels, high blood pressure, and obesity. Furthermore, the effects ofthe first stage conditions are additive; an increase in the number ofconditions causes an increase in the risk of developing more seriousdiseases on the Syndrome X continuum.

A patient who begins the Syndrome X continuum risks spiraling into amaze of increasingly deadly diseases. The next stages of the Syndrome Xcontinuum lead to overt diabetes, kidney failure, and heart failure,with the possibility of stroke and heart attack at any time. Syndrome Xis a dangerous continuum, and preventative medicine is the best defense.Diseases are currently most easily diagnosed in their later stages, butcontrolling them at a late stage is extremely difficult. Diseaseprevention is much more effective at an earlier stage.

Subsequent to the isolation of particular disease state marker sequencesas taught by the instant invention, the promulgation of various forms ofrisk-assessment tests are contemplated which will allow physicians toidentify asymptomatic patients before they suffer an irreversible eventsuch as diabetes, kidney failure, and heart failure, and enableeffective disease management and preventative medicine. Additionally,the specific diagnostic tests which evolve from this methodology providea tool for rapidly and accurately diagnosing acute Syndrome X eventssuch as heart attack and stroke, and facilitate treatment.

Accordingly, it is an objective of the instant invention to define adisease specific marker sequence which is useful in evidencing andcategorizing at least one particular disease state.

It is another objective of the instant invention to evaluate samplescontaining a plurality of biopolymers for the presence of diseasespecific marker sequences which evidence a link to at least one specificdisease state.

It is a further objective of the instant invention to elucidateessentially all biopolymeric moieties contained therein, wherebyparticularly significant moieties may be identified.

It is a further objective of the instant invention provide at least onepurified antibody which is specific to said disease specific markersequence.

It is yet another objective of the instant invention to teach amonoclonal antibody which is specific to said disease specific markersequence.

It is a still further objective of the invention to teach polyclonalantibodies raised against said disease specific marker.

It is yet an additional objective of the instant invention to teach adiagnostic kit for determining the presence of said disease specificmarker.

It is a still further objective of the instant invention to teachmethods for characterizing disease state based upon the identificationof said disease specific marker.

Other objectives and advantages of this invention will become apparentfrom the following description taken in conjunction with theaccompanying drawings wherein are set forth, by way of illustration andexample, certain embodiments of this invention. The drawings constitutea part of this specification and include exemplary embodiments of thepresent invention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation of derived data which characterizes a diseasespecific marker having a particular sequence useful in evidencing andcategorizing at least one particular state;

FIG. 2 is the characteristic profile derived via SELDI/TOF MS of thedisease specific marker of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

Serum samples from individuals were analyzed using Surface EnhancedLaser Desorption Ionization (SELDI) using the Ciphergen PROTEINCHIPsystem. The chip surfaces included, but were not limited to IMAC-3-Ni,SAX2 surface chemistries, gold chips, and the like.

Preparatory to the conduction of the SELDI MS procedure, variouspreparatory steps were carried out in order to maximize the diversity ofdiscernible moities educable from the sample. Utilizing a type ofmicro-chromatographic column called a C18-ZIPTIP available from theMillipore company, the following preparatory steps were conducted.

1. Dilute sera in sample buffer;

2. Aspirate and dispense ZIP TIP in 50% Acetonitrile;

3. Aspirate and dispense ZIP TIP in Equilibration; solution;

4. Aspirate and Dispense in serum sample;

5. Aspirate and Dispense ZIP TIP in Wash solution;

6. Aspirate and Dispense ZIP TIP in Elution Solution.

Illustrative of the various buffering compositions useful in the presentinvention are:

Sample Buffers (various low pH's): Hydrochloric acid (HCl), Formic acid,Trifluoroacetic acid (TFA), Equilibration Buffers (various low pH's):HCl, Formic acid, TFA;

Wash Buffers (various low pH's): HCl, Formic acid, TFA;

Elution Solutions (various low pH's and % Solvents): HCl, Formic acid,TFA;

Solvents: Ethanol,Methanol, Acetonitrile. Spotting was then performed,for example upon a Gold Chip in the following manner:

1. spot 2 ul of sample onto each spot

2. let sample partially dry

3. spot 1 ul of matrx, and let air dry.

HiQ Anion Exchange Mini Column Protocol

1. Dilute sera in sample/running buffer;

2. Add HiQ resin to column and remove any air bubbles;

3. Add Uf water to aid in column packing;

4. Add sample/running buffer to equilibrate column;

5. Add diluted sera;

6. Collect all the flow through fraction in Eppendorf tubes until levelis at resin;

7. Add sample/running buffer to wash column;

8. Add elusion buffer and collect elusion in Eppendorf tubes.

Illustrative of the various buffering compositions useful in thistechnique are:

Sample/Running buffers: including but not limited to Bicine buffers ofvarious molarities, pH's, NaCl content, Bis-Tris buffers of variousmolarities, pH's, NaCl content, Diethanolamine of various molarities,pH's, NaCl content, Diethylamine of various molarities, pH's, NaClcontent, Imidazole of various molarities, pH's, NaCl content, Tricine ofvarious molarities, pH's, NaCl content, Triethanolamine of variousmolarities, pH's, NaCl content, Tris of various molarities, pH's, NaClcontent.

Elution Buffer: Acetic acid of various molarities, pH's, NaCl content,Citric acid of various molarities, pH's, NaCl content, HEPES of variousmolarities, pH's, NaCl content, MES of various molarities, pH's, NaClcontent, MOPS of various molarities, pH's, NaCl content, PIPES ofvarious molarities, pH's, NaCl content, Lactic acid of variousmolarities, pH's, NaCl content, Phosphate of various molarities, pH's,NaCl content, Tricine of various molarities, pH's, NaCl content.

Chelating Sepharose Mini Column

1. Dilute Sera in Sample/Running buffer;

2. Add Chelating Sepharose slurry to column and allow column to pack;

3. Add UF water to the column to aid in packing;

4. Add Charging Buffer once water is at the level of the resin surface;

5. Add UF water to wash through non bound metal ions once charge bufferwashes through;

6. Add running buffer to equilibrate column for sample loading;

7. Add diluted serum sample;

8. Add running buffer to wash unbound protein;

9. Add elution buffer and collect elution fractions for analysis;

10. Acidify each elution fraction.

Illustrative of the various buffering compositions useful in thistechnique are: Sample/Running buffers including but not limited toSodium Phosphate buffers at various molarities and pH's;

Charging buffers including but not limited to Nickel Chloride, NickelSulphate, Copper II Chloride, Zinc Chloride or any suitable metal ionsolution;

Elution Buffers including but not limited to Sodium phosphate buffers atvarious molarities and pH's containing various molarities of EDTA and/orImidazole.

HiS Cation Exchange Mini Column Protocol

1. Dilute sera in sample/running buffer;

2. Add HiS resin to column and remove any air bubbles;

3. Add Uf water to aid in column packing;

4. Add sample/running buffer to equilibrate column for sample loading;

5. Add diluted sera to column;

6. Collect all flow through fractions in Eppendorf tubes until level isat resin.

7. Add sample/running buffer to wash column.

8. Add elusion buffer and collect elusion in Eppendorf tubes.

Illustrative of the various buffering compositions useful in thistechnique are:

Sample/Running buffers: including but not limited to Bicine buffers ofvarious molarities, pH's, NaCl content, Bis-Tris buffers of variousmolarities, pH's, NaCl content, Diethanolamine of various molarities,pH's, NaCl content, Diethylamine of various molarities, pH's, NaClcontent, Imidazole of various molarities, pH's, NaCl content, Tricine ofvarious molarities, pH's, NaCl content, Triethanolamine of variousmolarities, pH's, NaCl content, Tris of various molarities, pH's, NaClcontent.

Elution Buffer: Acetic acid of various molarities, pH's, NaCl content,Citric acid of various molarities, pH's, NaCl content, HEPES of variousmolarities, pH's, NaCl content, MES of various molarities, pH's, NaClcontent, MOPS of various molarities, pH's, NaCl content, PIPES ofvarious molarities, pH's, NaCl content, Lactic acid of variousmolarities, pH's, NaCl content, Phosphate of various molarities, pH's,NaCl content, Tricine of various molarities, pH's, NaCl content.

The procedure for profiling serum samples is described below:

Following the preparatory steps illustrated above, various methods foruse of the PROTEINCHIP arrays, available for purchase from CiphergenBiosystems (Palo Alto, Calif.), may be practiced. Illustrative of onesuch method is as follows.

The first step involved treatment of each spot with 20 ml of a solutionof 0.5 M EDTA for 5 minutes at room temperature in order to remove anycontaminating divalent metal ions from the surface. This was followed byrinsing under a stream of ultra-filtered, deionized water to remove theEDTA. The rinsed surfaces were treated with 20 ml of 100 mM Nickelsulfate solution for 5 minutes at room temperature after which thesurface was rinsed under a stream of ultra-filtered, deionized water andallowed to air dry.

Serum samples (2 ml) were applied to each spot (now “charged” with themetal-Nickel) and the PROTEINCHIP was returned to the plastic containerin which it was supplied. A piece of moist KIMWIPE was placed at thebottom of the container to generate a humid atmosphere. The cap on theplastic tube was replaced and the chip allowed to incubate at roomtemperature for one hour. At the end of the incubation period, the chipwas removed from the humid container and washed under a stream ofultra-filtered, deionized water and allowed to air dry. The chipsurfaces (spots) were now treated with an energy-absorbing molecule thathelps in the ionization of the proteins adhering to the spots foranalysis by Mass Spectrometry. The energy-absorbing molecule in thiscase was sinapinic acid and a saturated solution prepared in 50%acetonitrile and 0.05% TFA was applied (1 ml) to each spot. The solutionwas allowed to air dry and the chip analyzed immediately using MS(SELDI).

Serum samples from patients suffering from a variety of disease stateswere analyzed using one or more protein chip surfaces, e.g. a gold chipor an IMAC nickel chip surface as described above and the profiles wereanalyzed to discern notable sequences which were deemed in some wayevidentiary of at least one disease state.

In order to purify the disease specific marker and further characterizethe sequence thereof, additional processing was performed.

For example, Serum (20 ml) was (diluted 5-fold with phosphate bufferedsaline) concentrated by centrifugation through a YM3 MICROCON spinfilter (Amicon) for 20 min at 10,000 RPM at 4° C. in a BeckmanMICROCENTRIFuge R model bench top centrifuge. The filtrate was discardedand the retained solution, which contained the two peptides of interest,was analyzed further by tandem mass spectrometry to deduce their aminoacid sequences. Tandem mass spectrometry was performed at the Universityof Manitoba's (Winnipeg, Manitoba, Canada) mass spectrometry laboratoryusing the procedures that are well known to practitioners of the art.

As a result of these procedures, the disease specific markerADSGEGDFLAEGGGVR was found. This marker is characterized as a AlphaFibrinogen having a molecular weight of 1518 daltons. The characteristicprofile of the marker is set forth in FIG. 2. As easily deduced from thedata set forth in FIG. 1, this marker is indicative of myocardialinfarction.

In accordance with various stated objectives of the invention, theskilled artisan, in possession of the specific disease specific markeras instantly disclosed, would readily carry out known techniques inorder to raise purified biochemical materials, e.g. monoclonal and/orpolyclonal antibodies, which are useful in the production of methods anddevices useful as point-of-care rapid assay diagnostic or riskassessment devices as are known in the art.

The specific disease markers which are analyzed according to the methodof the invention are released into the circulation and may be present inthe blood or in any blood product, for example plasma, serum, cytolyzedblood, e.g. by treatment with hypotonic buffer or detergents anddilutions and preparations thereof, and other body fluids, e.g. CSF,saliva, urine, lymph, and the like. The presence of each marker isdetermined using antibodies specific for each of the markers anddetecting specific binding of each antibody to its respective marker.Any suitable direct or indirect assay method may be used to determinethe level of each of the specific markers measured according to theinvention. The assays may be competitive assays, sandwich assays, andthe label may be selected from the group of well-known labels such asradioimmunoassay, fluorescent or chemiluminescence immunoassay, orimmunoPCR technology. Extensive discussion of the known immunoassaytechniques is not required here since these are known to those ofskilled in the art. See Takahashi et al. (Clin Chem 1999;45(8):1307) forS100B assay.

A monoclonal antibody specific against the disease marker sequenceisolated by the present invention may be produced, for example, by thepolyethylene glycol (PEG) mediated cell fusion method, in a mannerwell-known in the art.

Traditionally, monoclonal antibodies have been made according tofundamental principles laid down by Kohler and Milstein. Mice areimmunized with antigens, with or without, adjuvants. The splenocytes areharvested from the spleen for fusion with immortalized hybridomapartners. These are seeded into microtitre plates where they can secreteantibodies into the supernatant that is used for cell culture. To selectfrom the hybridomas that have been plated for the ones that produceantibodies of interest the hybridoma supernatants are usually tested forantibody binding to antigens in an ELISA (enzyme linked immunosorbentassay) assay. The idea is that the wells that contain the hybridoma ofinterest will contain antibodies that will bind most avidly to the testantigen, usually the immunizing antigen. These wells are then subclonedin limiting dilution fashion to produce monoclonal hybridomas. Theselection for the clones of interest is repeated using an ELISA assay totest for antibody binding. Therefore, the principle that has beenpropagated is that in the production of monoclonal antibodies thehybridomas that produce the most avidly binding antibodies are the onesthat are selected from among all the hybridomas that were initiallyproduced. That is to say, the preferred antibody is the one with highestaffinity for the antigen of interest.

There have been many modifications of this procedure such as using wholecells for immunization. In this method, instead of using purifiedantigens, entire cells are used for immunization. Another modificationis the use of cellular ELISA for screening. In this method instead ofusing purified antigens as the target in the ELISA, fixed cells areused. In addition to ELISA tests, complement mediated cytotoxicityassays have also been used in the screening process. However,antibody-binding assays were used in conjunction with cytotoxicitytests. Thus, despite many modifications, the process of producingmonoclonal antibodies relies on antibody binding to the test antigen asan endpoint.

The purified monoclonal antibody is utilized for immunochemical studies.

Polyclonal antibody production and purification utilizing one or moreanimal hosts in a manner well-known in the art can be performed by askilled artisan.

Another objective of the present invention is to provide reagents foruse in diagnostic assays for the detection of the particularly isolateddisease specific marker sequences of the present invention.

In one mode of this embodiment, the marker sequences of the presentinvention may be used as antigens in immunoassays for the detection ofthose individuals suffering from the disease known to be evidenced bysaid marker sequence. Such assays may include but are not limited to:radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), “sandwich”assays, precipitin reactions, gel diffusion immunodiffusion assay,agglutination assay, fluorescent immunoassays, protein A or Gimmunoassays and immunoelectrophoresis assays.

According to the present invention, monoclonal or polyclonal antibodiesproduced against the disease specific marker sequence of the instantinvention are useful in an immunoassay on samples of blood or bloodproducts such as serum, plasma or the like, spinal fluid or other bodyfluid, e.g. saliva, urine, lymph, and the like, to diagnose patientswith the characteristic disease state linked to said marker sequence.The antibodies can be used in any type of immunoassay. This includesboth the two-site sandwich assay and the single site immunoassay of thenon-competitive type, as well as in traditional competitive bindingassays.

Particularly preferred, for ease and simplicity of detection, and itsquantitative nature, is the sandwich or double antibody assay of which anumber of variations exist, all of which are contemplated by the presentinvention. For example, in a typical sandwich assay, unlabeled antibodyis immobilized on a solid phase, e.g. microtiter plate, and the sampleto be tested is added. After a certain period of incubation to allowformation of an antibody-antigen complex, a second antibody, labeledwith a reporter molecule capable of inducing a detectable signal, isadded and incubation is continued to allow sufficient time for bindingwith the antigen at a different site, resulting with a formation of acomplex of antibody-antigen-labeled antibody. The presence of theantigen is determined by observation of a signal which may bequantitated by comparison with control samples containing known amountsof antigen.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementherein described and shown. It will be apparent to those skilled in theart that various changes may be made without departing from the scope ofthe invention and the invention is not to be considered limited to whatis shown and described in the specification and drawings/figures.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned, as well as those inherent therein. Theoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method for diagnosing myocardial infarction comprising: (a)obtaining a sample from a patient; (b) conducting mass spectrometricanalysis on said sample in a manner effective to maximize elucidation ofdiscernible peptide fragments contained therein; and (c) comparing massspectrum profiles of a peptide consisting of SEQ ID NO:1 to massspectrum profiles of peptides elucidated from said sample; whereinrecognition of a mass spectrum profile in the sample displaying thecharacteristic profile of the mass spectrum profile for the peptideconsisting of SEQ ID NO:1 is diagnostic for myocardial infarction. 2.The method of claim 1, wherein said sample is an unfractionated bodyfluid or a tissue sample.
 3. The method of claim 1, wherein said sampleis selected from the group consisting of blood, blood products, urine,saliva, cerebrospinal fluid, and lymph.
 4. The method of claim 1,wherein said mass spectrometric analysis is Surface Enhanced LaserDesorption Ionization (SELDI) mass spectrometry (MS).
 5. The method ofclaim 1, wherein said patient is a human.
 6. A myocardial infarctiondiagnostic kit comprising: (a) a peptide consisting of SEQ ID NO:1 and(b) an antibody that binds to said peptide in a sample from a patient.7. The diagnostic kit of claim 6, wherein said antibody is immobilizedon a solid support.
 8. The diagnostic kit of claim 6, wherein saidantibody is labeled.